1. Field of the Invention
The invention relates to a method for generating an at least two-dimensional image of at least a part of a sample. The optical devices can be, for example, telescopes, microscopes, material-testing devices and other analyzing devices. The origin of the light is inconsequential, so that the invention can serve, for example, in conjunction with scintillation for the detection of ionizing radiation. However, the invention is preferably used for the detection of fluorescence.
2. Related Art
Microscopic examinations with fluorescent dyes result in many instances in heterogeneous distributions of concentration in the samples to be examined. This can concern desired attachments such as are generated, for example, for the structured marking in cellular samples or tissue samples by directly applied—for example, specific antibody dyes—or expressed fluorescent dyes such as, for example, GFP. Also, additional undesired inhomogeneities are frequently included, for example, by autofluorescence and heavily fluorescing tissue inclusions.
Heavily heterogeneous fluorescence distributions in a microscopic image have the result, like great differences of brightness in conventional photography, that when using the dynamic range of the detector in an individual photograph only a part of the image information can be detected. In order to be able to resolve contrast details in bright image areas (with high average light intensities), the detector must not be saturated. To this end the photographing time (also designated as exposure time or integration time) must be selected to be short. However, as a consequence dark image areas (with low average light intensities) are recorded only with insufficient signal-to-noise ratio (SNR), that is, with poor contrast. Inversely, a longer exposure time does make possible the contrast-rich photographing of dark image areas but results in an overcontrol (low contrast) of the bright image areas.
Dark as well as bright image areas can be separately measured with a good SNR ratio by a multiple photographing of individual images with different exposure times. They can then be combined to a total image with expanded dynamics (Engl. “High Dynamic Range Imaging”; HDRI; A. Bell et al.: “High Dynamic Range Images as a Basis for Detection of Argyrophilic Nucleolar Organizer Regions Under Varying Stain Intensities”, Proceedings of IEEE International Conference on Image Processing, ICIP 2006, 2541-2544). Instead of different exposure times, in the case of fluorescence different excitation intensities can be used.
It is known from U.S. Pat. No. 7,859,673 B2, whose disclosed content is incorporated herein in its entirety, to use a detector module in a laser scanning microscope (LSM) in which module a light signal strikes an individual optoelectronic converter, where it is converted into an electrical signal that is subsequently divided into several parallel evaluation channels. A signal evaluation is carried out in each evaluation channel, which evaluation is different from the signal evaluations for the other evaluation channels, and that generates a result signal. One or more of the result signals are selected and outputted using a given, variably adjustable selection criterion. Thus, several individual images with different acquisition methods can be taken by simply switching the evaluation channel used for image generation.
However, the sequential acquisition of several individual images is expensive and slow. In particular, it is problematic in the observation of fluorescence since the intensity of the fluorescence changes between the acquisitions by the fading of the fluorophones. Especially in the case of living samples the multiple acquisition results in a high beam load, especially in the case of scanning methods such as in laser scanning microscopy. In addition, the sequential acquisition is not suitable for the observation of dynamic operating sequences.
DE 102 53 108 A1 describes a device that realizes an electrical signal division onto two evaluation channels by means of a high pass/low pass combination. Here, only a part of the detector information (that is, of the electrical signal) is made available for each detection channel. This can limit the possible evaluation methods or lead to increased expenses for the reduction of error recognition. For example, the average steady component is a function of the counting frequency in the high pass branch. In addition, the determination of the boundary frequencies of the high pass/low pass combination necessary for the development time renders difficult the adaptation to new applications or detection methods during the service life of the device. Moreover, the high pass/low pass combination leads to non-linear amplitudes and phase distortions that can have a disadvantageous effect in particular in frequency area methods (Engl. “frequency domain”).
Alternatively to the sequential acquisition of individual images, EP 1 761 071 A1, for example, teaches the simultaneous acquisition of individual images with different contrast ranges by asymmetric optical beam division. However, this obligatorily requires a division of the light signal onto several channels and several optoelectronic detectors that represent additional noise sources. The SNR is further reduced on account of the lesser signal strength in the individual channels due to the division.
The invention is based on the problem of indicating a method and an optical device of the initially cited type that make it possible to generate images with a broader dynamic range with low cost in a short time.